Abstract

AIM:

To study the protein C activation system in human liver myofibroblasts, and the effects of activated protein C (APC) on these cells.

METHODS:

Human liver myofibroblasts were obtained by outgrowth. Expression of protease activated receptor 1 (PAR-1), endothelial protein C receptor (EPCR) and thrombomodulin (TM) was analyzed by flow cytometry. Extracellular signal-regulated kinase (ERK)1/2 activation was assessed by Western blotting using anti-phospho-ERK antibodies. Collagen synthesis was studied with real-time reverse transcription-polymerase chain reaction (RT-PCR). Activation of protein C was studied by incubating liver myofibroblasts with zymogen protein C in the presence of thrombin and detecting the generation of APC with a colorimetric assay using a peptide substrate.

RESULTS:

Primary cultures of human liver myofibroblasts expressed EPCR on their surface, together with PAR-1 and TM. This receptor system was functional since exposure of myofibroblasts to APC induced ERK1/2 phosphorylation in a dose- and time-dependent manner. Furthermore, APC significantly upregulated the expression of collagen mRNA, as shown by real-time RT-PCR. Collagen upregulation was controlled through the ERK pathway as it was inhibited when using the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor PD98059. Finally, using a cell-based colorimetric assay, we showed that intact myofibroblasts converted protein C into APC in the presence of thrombin.

CONCLUSION:

These data suggest that APC is a new modulator of liver myofibroblast activity and contributes to the pathophysiology of chronic liver diseases.

Effect of activated protein C (APC) on extracellular signal-regulated kinase (ERK) phosphorylation. A: Dose-response of APC. Cells were exposed for 10 min to 27 nmol/L thrombin (Thr), 100 nmol/L nonactivated protein C (PC), or APC. ERK phosphorylation was assessed using Western blotting. The upper panel shows a blot labeled with an antibody to phospho-ERK, and the lower panel shows the same blot re-hybridized with an antibody to total ERK. The graph shows the quantification of p42 phosphorylation in three similar independent experiments. Results are shown as mean ± SE. Phospho-p42 signals were normalized first to total p42 signals, and the results were expressed as a percentage of the non-stimulated control. Similar results were obtained when quantitating p44 phosphorylation; B: Kinetics of APC induced phosphorylation of ERK. Cells were exposed to 100 nmol/L APC. The upper panel shows a blot labeled with an antibody to phospho-ERK, and the lower panel shows the same blot re-hybridized with an antibody to total ERK. The graph shows the quantification of p44 phosphorylation in three similar independent experiments. Phospho-p44 signals were normalized first to total p44 signals, and the results were expressed as a percentage of the non-stimulated control. Similar results were obtained when quantitating p42 phosphorylation; C: APC effect is not due to contaminating thrombin. Cells were exposed for 10 min to 27 nmol/L Thr or 100 nmol/L APC. They were pretreated with or without hirudin. The graph shows the quantification of combined ERK1/ERK2 phosphorylation in four similar independent experiments; D: APC signals through PAR-1. Cells were exposed for 10 min to 27 nmol/L Thr or 100 nmol/L APC. They were preincubated with the indicated antibodies. The graph shows the quantification of combined ERK1/ERK2 phosphorylation in three similar independent experiments.

APC upregulates collagen transcripts. A: Cells were exposed for 24 h to 100 nmol/L APC or 1 ng/mL transforming growth factor-β1. Collagen transcripts were measured by real-time reverse transcription-polymerase chain reaction and normalized to those of the RPL0 gene. Results are expressed as fold increase over control values and are the mean ± SE of five experiments; B: Cells were exposed for 6 h to 100 nmol/L APC, in the presence or absence of the mitogen-activated protein kinase (MAPK) inhibitor PD98059 (50 μmol/L), before measuring collagen transcripts. Results are the mean ± SE of three experiments.

Human liver myofibroblasts can generate APC from PC. Supernatants were collected and incubated with an APC chromogenic substrate. The graph shows the optical density at 405 nmol/L, which resulted from cleavage of the substrate, which was proportional to the amount of APC generated. It represents the mean of two separate experiments.